An enzyme exhibits not only high catalytic activity but also specificity. Such specificity includes stereospecificity as well as substrate specificity and reaction specificity. The stereospecificity of an enzyme is almost absolute although there are some exceptions.
Recent studies have become more and more dependent on high precision technologies. In this context, it becomes increasingly important to use optically active isomers in the fields of pharmaceuticals, pesticides, feed, flavor, and others. Technologies to isolate specific optical isomers are exceedingly important because their physiological activities are sometimes quite distinct to each other. Thus, how to isolate (synthesize or resolve) optically pure enantiomers is an industrially important objective.
D-amino acids are non-proteinaceous amino acids and have been known to occur naturally in small cyclic peptides, peptide glycans in bacterial cell walls, and peptidic antibiotics over a long time. Recently, D-amino acids have been revealed to exist as a bound form in constituents of neuropeptides, dental enamel proteins, and proteins in crystalline lenses and in the brain, and thus many studies have been carried out to elucidate the physiologic significances of D-amino acids and enzymatic methods for synthesizing D-amino acids.
D-amino acids are widely used as important intermediates in the synthesis of pesticides, pharmaceuticals, and such. D-tryptophan is used as an intermediate in the synthesis of an agent to treat erectile dysfunction.
Previously established methods for producing D-amino acids include, for example, the following methods:
(1) A method that comprises chemically hydrolyzing 5-substituted hydantoin to produce a corresponding DL-amino acids and isolating a D-amino acid by optical resolution. (2) A method that comprises contacting 5-substituted hydantoin with a microorganism, its culture medium or its processed product, which is capable of producing an optically active D-N-carbamyl amino acid from 5-substituted hydantoin, and preparing a D-amino acid using sodium nitrite or D-carbamoylase (WO 94/03613). An alternative method comprises contacting 5-substituted hydantoin with a microorganism, its culture medium, or its processed product, which is capable of directly producing a D-amino acid from 5-substituted hydantoin. The following microorganisms can be used in this method:
The genus Pseudomonas (Unexamined Published Japanese Patent Application (JP-A) No. Sho 54-2398)
The genus Moraxella (JP-A No. Sho 54-89089)
The genus Hansenula (JP-A No. Sho 61-177991)
(3) A method that comprises reacting a DL-amino acid with a microorganism, its culture medium, or its processed product, which is capable of decomposing the L form, and recovering the residual D-amino acid (JP-A No. Hei 09-75097).
(4) A method that comprises contacting L-aminoacylase with an N-acetyl-DL-amino acid to hydrolyze, N-acetyl-L-amino acid which is one enantiomer of the DL-amino acid, recovering the residual N-acetyl-D-amino acid, and chemically hydrolyzing the N-acetyl -D-amino acid to produce a D-amino acid (Methods in Enzymology. 3, 554).
(5) A method that comprises contacting DL-amino acid amide with cells or a processed product of a microorganism having D-amidase or D-amidase activity that selectively hydrolyzes only the D form of DL-amino acid amide, to produce ad-amino acid (JP-A No. Hei 02-234678), or method that comprises contacting DL-amino acid amide with cells or a processed product of a microorganism having L-amidase or L-amidase activity that selectively hydrolyzes only the L form of DL-amino acid amide and chemically hydrolyzing the residual D-amino acid amide to produce a D-amino acid (JP-A No. Sho 57-013000).
(6) A method that comprises producing a D-amino acid from a corresponding α-keto acid by contacting D-amino acid transaminase with the α-keto acid in the presence of a D-amino acid as an amino group donor (JP-A No. Sho 62-205790).
(7) A method that comprises contacting DL-tryptophan with cells or a processed product of a microorganism having tryptophanase or tryptophanase activity that selectively decomposes L-tryptophan, and recovering the residual D-tryptophan (JP-A No. Hei 11-042097).
However, the methods described above have various problems, including high cost of materials, complicated processes, and low yields. Thus, with these methods, it is quite difficult to produce D-tryptophan in high yield and with low cost. In contrast to the methods described above, the following D-amino acid production method is known.
(8) A method that comprises contacting D-aminoacylase with an N-acetyl-DL-amino acid to hydrolyze only an N-acetyl-D-amino acid which is one enantiomer of the DL-amino acid, thereby producing a D-amino acid (JP-A No. Sho 53-059092).
With this method, D-tryptophan can be produced by a single-step enzymatic reaction using N-acetyl-DL-tryptophan as a starting material, which is synthesized from inexpensive L-tryptophan and acetic anhydride.
Examples of known microorganisms that produce D-aminoacylase include the following:
The genus Pseudomonas: 
Pseudomonas sp. AAA6029 (Chem. Pharm. Bull., 26, 2698(1978));
Pseudomonas sp. 1158 (J. Antibiot., 33, 550 (1980));
Pseudomonas sp. 5f-1 (Appl. Environ. Microbiol. 57, 2540(1991));
The genus Streptomyces: 
Streptomyces olivaceus (Argric. Biol. Chem., 42, 107 (1978));
Streptomyces olivaceus 62-3 (Argric. Biol. Chem., 44, 1089(1980));
Streptomyces olivaceus S-62 (JP-A No. Sho 53-59092);
Streptomyces thermonitrificans CS5-9 (JP-A No. 2002-45179);
The genus Alcaligenes: 
Alcaligenes denitrificans subsp. denitrificans DA181 (Appl. Environ. Microbiol., 54, 984(1988));
Alcaligenes faecalis DA1 (Appl. Environ. Microbiol., 57, 1259(1991));
Alcaligenes xylosoxidans subsp. xylosoxydans A-6 having an enzyme acting on acidic N-acyl-D-amino acids (FEBS, 289, 44(1991), Biosci. Biotech. Biochem., 57, 1145(1993)) and an enzyme acting on neutral N-acyl-D-amino acids (Biosci. Biotech. Biochem., 57, 1149(1993));
Alcaligenes denitrificans subsp. xylosoxydans MI-4 (J. Ferment. Bioeng., 71, 79 (1991));
Alcaligenes sp. (WO 00/23598);
Others:
Stenotrophomonas maltophilia (J. Industrial Microbiol. Biotechnol., 21, 296(1998))
Arthrobacter hydrocarboglutamicus (JP-A No. Hei 11-113592)
Amycolatopsis orientaris (JP-A No. Hei 11-98982)
Sebekia benihana (JP-A No. Hei 11-318442)
Hypomyces mycophilus (JP-A No. 2000-41684)
Rhodococcus rhodochrous 
Pimelobacter simplex (JP-A No. Hei 06-22789),
Methylobacterium mesophilicum 
Nocardioides thermolilacinus (WO 02/061077)
Trichoderma harzianum 
The enzymes derived from the genus Stenotrophomonas, the genus Rhodococcus, and the genus Pimelobacter, as listed above, have not been purified, and thus the properties of these enzymes still remain to be clarified. Although the enzyme derived from the genus Arthrobacter has been purified, its properties still remain unknown.
N-acyl-D-glutamic acid amidohydrolase derived from the genus Pseudomonas, and N-acyl-D-glutamic acid deacetylase, N-acyl-D-aspartic acid amide hydrolase, and D-aminoacylase derived from the genus Alcaligenes A-6 strain have been reported to be all inactive to N-acetyl-D-tryptophan.
According to references, the enzymes derived from the genus Pseudomonas, the genus Streptomyces, the genus Trichoderma, and the genus Amycolatopsis showed as low as 10 U/mg or lower of activity to N-acetyl-D-tryptophan and no activity to N-acetyl-D-tryptophan.
Furthermore, the activities of the enzymes derived from another strain DA1 belonging to the genus Alcaligenes, from the genus Hypomyces, and from the genus Sebekia are 100 U/mg or lower. On the other hand, the activity of the enzyme derived from another strain DA181 of the genus Alcaligenes has been reported to be as high as about 600 U/mg for N-acetyl-D-tryptophan. However, its stereoselectivity is not strict because this enzyme also shows the activity of about 11 U/mg to N-acetyl-L-tryptophan.
A novel D-aminoacylase derived from Alcaligenes sp. (WO00/23598) has been reported to act on N-acetyl-D-tryptophan. However the report shows only that the enzyme has the hydrolytic activity to 25 mM N-acetyl-DL-tryptophan and 10 mM N-acetyl-D-tryptophan. This D-aminoacylase is reportedly a novel enzyme capable of catalyzing the hydrolysis of 10 g/l N-acetyl-D-tryptophan.
On the other hand, the known D-aminoacylase derived from Alcaligenes denitrificans subsp. xylosoxydans MI-4 strain was found to be able to produce about 2.5 g/l D-tryptophan from the substrate, 150 g/l N-acetyl-DL-tryptophan, which corresponds to 75 g/l N-acetyl-D-tryptophan, as shown in FIG. 1. Thus, the enzyme was confirmed to have a sufficiently high activity to hydrolyze such a high concentration of the substrate.
It has been reported that the D-aminoacylases derived from Methylobacterium mesophilicum and Nocardioides thermolilacinus act on N-acetyl-D-tryptophan, and the catalytic reaction are hardly inhibited by the substrate even at concentrations as high as 100 g/l. However, the report does not mention the competitive inhibition by N-acetyl-L-tryptophan for the two enzymes. In addition, no detailed information is available for the two enzymes, and particularly, the enzyme derived from Nocardioides thermolilacinus has been neither purified nor characterized so far.
It was reported that when E. coli transformed with DNA containing a D-aminoacylase gene derived from Methylobacterium mesophilicum was incubated with 100 g/l N-acetyl-DL-tryptophan, D-tryptophan was produced in yield of about 90% from the substrate, N-acetyl-D-tryptophan (WO02/061077). However, there is no report on the production of D-tryptophan from higher concentrations of the substrate. For industrial production of D-tryptophan, it is desirable to hydrolyze a high concentration of the substrate, N-acetyl-DL-tryptophan, into D-tryptophan .